To define the molecular pathway of CTL-mediated cytotoxicity, we previously tested the possibility that precursors of caspases (endogenous "death proteases") are the physiologically relevant granzyme substrates leading to target lysis. By using two types of caspase inhibitor, we found that caspases were required for the FasL/Fas cytotoxicity pathway and also for nuclear damage by the granule exocytosis pathway, but were not required for target lysis by the latter pathway. We have now tested whether other pre-lytic non-nuclear target damage inflicted by the granule exocytosis pathway is caspase-dependent. Using flow cytometry and a lipid probe to mark target cells, we have found that target cell phosphatidyl serine is rapidly exposed on the target cell outer membrane in response to damage by CTL or NK cells using both the granule exocytosis and FasL/Fas pathways. PS exposure via the former pathway is not affected by peptide-FMK caspase inhibitors, while PS exposure via the latter is specifically and completely blocked. We have used DiOC5 to measure mitochondrial potential psi and found that both pathways of CTL-induced damage give a rapid loss of psi. Caspase inhibitors again block loss via the FasL/Fas pathway but not the granule exocytosis pathway. We have also examined target cells microscopically for the appearance of membrane blebs, and found that CTL induce rapid blebbing by both damage pathways. Again caspase inhibitors block blebbing induced by the FasL/Fas pathway but not the granule exocytosis pathway. Thus for three different non-nuclear measures of target damage shared by the two damage pathways, caspase inhibitors block the FasL/Fas pathway but not the granule exocytosis pathway. These findings support our previously proposed model that granzymes and caspases share common substrates leading to target cell death, while granzyme-induced nucelar damage occurs via caspases as previously proposed.